The prototypic arenavirus LCMV has proven to be a Rosetta stone for the investigation of the anti-viral host immune responses, and mechanisms of virus immune evasion and persistence. In addition, evidence indicates that LCMV, worldwide distributed, is a neglected human pathogen of clinical significance. Moreover, several other arenaviruses cause hemorrhagic fever (HF) disease in humans. The failure of the host innate immune response to control virus multiplication contributes to arenaviral-induced disease. We have shown that the nucleoprotein (NP) of LCMV and HF arenaviruses inhibits activation of the transcriptional factor IRF3 and production of Type I interferon (IFN-I), a key player in innate immunity. Our long-term goal is to understand the molecular bases and biological consequences of the IFN counteracting activity associated with arenavirus NP. To this end we propose the following specific aims: Aim 1. Define the genetic determinants of LCMV-NP responsible for its IFN antagonistic function. We have identified two C-terminal domains of LCMV-NP that are critical for the anti-IFN-I activity of NP. We hypothesize that specific residues within these two regions play a critical role on the IFN antagonistic function of NP, and that this function can be segregated from the role of NP in virus replication and production of infectious progeny. We propose to conduct a comprehensive mutation- function analysis of these regions to identify single amino acid mutations in NP that abrogate its anti-IFN function without affecting other roles of NP required for arenavirus multiplication. Aim 2. Define the mechanisms by which NP exerts its IFN antagonistic activity. We have shown that LCMV-NP counteracts the IFN response by inhibiting activation and nuclear translocation of IRF3. We hypothesize that LCMV-NP achieves this by interacting with host cell proteins involved in activation of IRF3. We will pursue proteomic approaches including affinity purification methods combined with mass spectrometry procedures to identify NP-interacting cellular proteins. We will use biochemical and genetic approaches, together with functional assays, to assess the contribution of identified NP-interacting cellular proteins to the IFN antagonistic activity of NP. Aim 3. Assess the contribution of the IFN antagonistic activity of NP to virus multiplication and pathogenesis during LCMV infection of its natural host, the mouse. We will generate recombinant LCM viruses (rLCMV/NP*) containing mutated forms of NP that retain wt activity in virus RNA synthesis and infectious particle formation but lost the ability to inhibit IRF3 activation and IFN production. We hypothesize that rLCMV/NP*-infected cells will exhibit higher levels of IRF3-inducible gene expression, including IFN production, compared to LCMV/WT-infected cells. We predict this to result in altered growth properties of rLCMV/NP* with consequences for the outcome of LCMV-host interaction during acute and persistent infection. PUBLIC HEALTH RELEVANCE Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and the worldwide-distributed prototypic arenavirus LCMV is being considered as a neglected human pathogen of clinical significance. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Evidence indicates that a failure of the host innate immune response to control virus multiplication plays a critical role in arenaviral- induced disease. We have shown that the nucleoprotein (NP) of LCMV and HF arenaviruses inhibits activation of the transcriptional factor IRF3 and production of Type I interferon (IFN), a key player in innate immunity. Our long-term goal is to understand the molecular bases and biological consequences of the IFN counteracting activity associated with arenavirus NP. This knowledge should fuel the development of better antiviral strategies to combat human pathogenic arenaviruses.